PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) cells maintain proliferative capacity despite a hypovascular, nutrient- poor microenvironment; this challenging microenvironment is established by cancer-associated fibroblasts (CAFs). PDAC CAFs are predominantly derived from pancreatic stellate cells (PSCs), lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype and generate a dense, desmoplastic stroma. CAF-derived components generate an oxygen- and nutrient-poor microenvironment to which PDAC cells must adapt. These adaptation mechanisms remain poorly understood, and may represent vulnerabilities for therapeutic intervention. Our recent results suggest that paracrine lipid flux from PSC-derived CAFs represents a novel mechanism by which the stroma provides energy and pro-proliferative signals to cancer cells to support PDAC growth. Specifically, we find that PDAC CAFs secrete abundant lysophosphatidylcholine (LPC), the preferred fatty acid scavenging substrate for Ras-transformed cells such as PDAC cells; the PDAC CAF secretome also contains high levels of lysophosphatidic acid (LPA), an established mitogen with multiple downstream effectors with roles in tumorigenesis. Our preliminary transcriptional and lipidomic data suggest that PSCs undergo a dramatic lipid metabolic shift in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated CAF state. These results raise the possibility that PDAC CAFs secrete specific lipid species that act in a paracrine manner to ?feed? the epithelial compartment and stimulate proliferation. We hypothesize that fibroblastic cells function via secreted lipids to promote epithelial cell proliferation and survival in the context of an inhospitable wound-healing response. This hypothesis will be tested with the following specific aims. Aim 1: Understand the effect of stroma-derived lipids on cancer cell lipid metabolism. A stable isotope tracing approach will be used to determine the extent of paracrine metabolic flux from stromal cells to cancer cells among secreted lipids, and alterations to PDAC cell metabolism in response to stroma-derived lipids will be assessed using established metabolic assays. Aim 2: Determine the role of stroma-derived lipids in PDAC cell growth control. Established in vitro culture and co-culture systems will be used to analyze regulation of mitogenic LPA effectors pathways and growth capacity in response to stroma-derived lipids. Aim 3: Define the significance of the LPC-Autotaxin-LPA axis in microenvironmental regulation of pancreatic cancer growth in vivo. LPA is generated from LPC by secreted enzyme Autotaxin; we find that LPC is secreted at high levels by PDAC CAFs, and that ATX is overexpressed in mouse and human PDAC. The LPC-Autotaxin-LPA axis will be interrogated both genetically and pharmacologically in a PDAC mouse model in vivo to determine its role in PDAC progression. These studies will improve our understanding of PDAC cell survival and growth mechanisms in the context of a nutrient-poor tumor microenvironment, potentially identifying a novel route for therapeutic intervention.